Generation of Influenza Virus Escape Variants against Neutralizing Antibodies

Take an influenza virus suspension, containing both wild-type and mutant strains.

A viral envelope glycoprotein termed hemagglutinin, or HA, binds to sialic acid on the host cell surface, mediating viral entry.

Add a neutralizing antibody in decreasing concentrations that binds to HA, blocking the sialic acid binding site.

Mutant strains, carrying mutations in the HA that help avoid antibody neutralization, are termed escape variants.

Inject the mixture into pathogen-free embryonated chicken eggs, delivering the viruses into the allantoic fluid, and incubate.

Antibody-neutralized viruses cannot enter the cells of the chorioallantoic membrane. Non-neutralized mutant viruses enter the cells, replicate, and are released into the allantoic fluid.

Harvest the virus population-containing allantoic fluid.

Introduce an HA-specific antibody in decreasing concentrations, which neutralizes the other strains except the escape variants.

Introduce chicken red blood cells or RBCs. Non-neutralized escape variants bind to the cells, causing the clumping of RBCs — termed hemagglutination, confirming escape variant generation.

Neutralizing antibodies that have HI activity are further analyzed by first preparing 4 dilutions of the antibody of interest in increasing concentrations in 1 times PBS in a volume of 100 microliters per dilution. Next, prepare a virus stock of 1 million plaque-forming units per milliliter in 1 times PBS in a 400 microliter volume.

Then, mix 100 microliters of 1 million plaque-forming units per milliliter of virus with 100 microliters of each antibody dilution or 100 microliters of 1 times PBS. Incubate the samples for one hour in a 37 degrees Celsius incubator with 5% carbon dioxide.

After vortexing briefly, inject 200 microliters of each mixture into specific pathogen-free embryonated chicken eggs. Then, incubate the eggs at 37 degrees Celsius without carbon dioxide for 40 to 44 hours. Following incubation, sacrifice the virus-infected infected embryonated eggs by placing them at 4 degrees Celsius for a minimum of 6 hours. Next, harvest the allantoic fluid from the eggs as previously described.

Finally, confirm the escape variants by performing the HI assay as described in the text protocol.